Impact of host demography and evolutionary history on endosymbiont molecular evolution: A test in carpenter ants (genus Camponotus) and their Blochmannia endosymbionts

Abstract Obligate endosymbioses are tight associations between symbionts and the hosts they live inside. Hosts and their associated obligate endosymbionts generally exhibit codiversification, which has been documented in taxonomically diverse insect lineages. Host demography (e.g., effective population sizes) may impact the demography of endosymbionts, which may lead to an association between host demography and the patterns and processes of endosymbiont molecular evolution. Here, we used whole‐genome sequencing data for carpenter ants (Genus Camponotus; subgenera Camponotus and Tanaemyrmex) and their Blochmannia endosymbionts as our study system to address whether Camponotus demography shapes Blochmannia molecular evolution. Using whole‐genome phylogenomics, we confirmed previous work identifying codiversification between carpenter ants and their Blochmannia endosymbionts. We found that Blochmannia genes have evolved at a pace ~30× faster than that of their hosts' molecular evolution and that these rates are positively associated with host rates of molecular evolution. Using multiple tests for selection in Blochmannia genes, we found signatures of positive selection and shifts in selection strength across the phylogeny. Host demography was associated with Blochmannia shifts toward increased selection strengths, but not associated with Blochmannia selection relaxation, positive selection, genetic drift rates, or genome size evolution. Mixed support for relationships between host effective population sizes and Blochmannia molecular evolution suggests weak or uncoupled relationships between host demography and Blochmannia population genomic processes. Finally, we found that Blochmannia genome size evolution was associated with genome‐wide estimates of genetic drift and number of genes with relaxed selection pressures.

Obligate endosymbionts not only share linked evolutionary histories with their hosts, but endosymbiont demography may also be impacted by host demography (Wernegreen, 2002). Because host and endosymbiont effective population sizes are (at least partially) intrinsically linked, host demographic history may influence the potential strength of selection and rate of genetic drift in endosymbiont genomes. Effective population size is linked with the potential strength of selection and in general, we may expect relaxed selection strengths in relatively smaller population sizes. Additionally, selection in endosymbiont genomes will be partially affected by host-level selection because endosymbiont fitness is partially linked with host fitness (Wernegreen, 2002). In addition to selection, rates of genetic drift are strongly linked with effective population sizes.
Asexual organisms-including bacterial endosymbionts-are also subject to accumulation and fixation of deleterious mutations due to their lack of recombination during reproduction (Moran, 1996;Muller, 1964;Pettersson & Berg, 2007). In a simulation study, Rispe and Moran (2000) showed that endosymbiont mutation fixation rate was higher in relatively smaller host populations. However, endosymbiont population sizes could also be decoupled from host population sizes. For example, endosymbiont transmission population bottlenecks (Mira & Moran, 2002) that vary in different host populations or species would lead to variance in the relationship between host and endosymbiont population sizes. Additionally, endosymbionts may be subject to within-host selection (Perreau et al., 2021) that could differentially change endosymbiont population sizes in different host populations. Despite the potential for host demography to shape endosymbiont molecular evolution, this topic has been largely unexplored in wild host and obligate endosymbiont systems.
The symbiotic relationship between carpenter ants (genus Camponotus Mayr, 1861) and their Blochmannia bacterial endosymbionts is an ideal system for investigating the influence of host demographic history on its associated endosymbionts. Camponotus is the second largest ant genus with over 2000 species grouped in 45 subgenera (AntWeb, 2022;Ward et al., 2016); these ants are common in woodlands across most of the world (Mackay, 2019;Wilson, 1976).
Camponotus belongs to the formicine tribe Camponotini that is composed of eight extant genera (Ward et al., 2016). Camponotines have maintained a relationship with Blochmannia for about 40 million years (Wernegreen et al., 2009); Blochmannia is a vertically transmitted, obligate intracellular bacterial symbiont (Ward et al., 2016) that was first recognized during the late 1800s (Blochmann, 1892).
Blochmannia are found in specialized cells (bacteriocytes) associated with host midgut tissue and found in the ovaries and oocytes of reproductive females (Ramalho et al., 2018;Wernegreen et al., 2009;Wolschin et al., 2004). Blochmannia provide amino acids to their hosts (Feldhaar et al., 2007), and, consistent with a long-term endosymbiosis, there is evidence that Camponotus and Blochmannia have histories of co-speciation because the evolutionary history of the symbionts reflects that of the ants (Degnan et al., 2004;Sauer et al., 2000;Wernegreen et al., 2009).

Several aspects of the Camponotus-Blochmannia relationship
have been studied in detail, including location of the symbionts in the host body (Kupper et al., 2016), transmission method (Ramalho et al., 2018), relative abundance and transcriptional variation across host developmental stages (Ramalho et al., 2017;Stoll et al., 2009), the effects of host development and reproduction on symbiont replication (Wolschin et al., 2004), and the endosymbiont's beneficial role in host nutrition (Feldhaar et al., 2007). One aspect of the Camponotus-Blochmannia relationship that remains poorly known is how host evolutionary and demographic histories affect endosymbiont molecular evolution. A few studies to date have examined the evolution of entire Blochmannia genomes; in Blochmannia vafer, there is some evidence of ongoing purifying selection (Williams & Wernegreen, 2012), and in comparative analyses across three Blochmannia genomes, gene loss patterns differ across lineages, suggestive of differential selective pressures in different host lineages (Williams & Wernegreen, 2015).
Here, we aimed to study how Camponotus demography and evolutionary history impacts molecular evolution in Blochmannia endosymbionts using data from seven Camponotus species (Figure 1; Table 1). Our study taxa include seven species from two subgenera in the genus Camponotus: Tanaemyrmex Ashmead, 1905 (N = 3 species) and Camponotus (N = 4 species). All the species included here are large ants (major workers >1 cm) associated with woodlands in western North America (Table 1; Table 2). Western North American species of the subgenus Tanaemyrmex tend to nest in the soil including soil under rocks or logs while the subgenus Camponotus tends to nest in decaying logs and stumps (Mackay, 2019). Carpenter ants are omnivorous; their feeding is comprised of opportunistic predation and foraging of animal and plant-derived resources (Mackay, 2019) and they are thought to have nitrogen-poor diets (Moreau, 2020). F I G U R E 1 Phylogenetic congruence of host ants and their Blochmannia endosymbionts. Branch labels indicate proportion of trees supporting this phylogenetic hypothesis. The ASTRAL species tree topologies were identical to these phylogenies and exhibited 100% quartet support for every relationship. Host trees were rooted with the Cataglyphis nigra sample. The Blochmannia tree was midpoint rooted. Orange branches in the Blochmannia tree indicate branches that vary between the host and endosymbiont phylogenies. Ant photos by JCG and JDM    Figure 1).

| Camponotus de novo genome assembly and annotation
As of January 2022, the only available genome of a Camponotus species is that of Camponotus (Myrmothrix) floridanus (Buckley, 1866) (Genome Accession: GCA_003227725.1 Cflo_v7.5). We decided to assemble the genome of a member of the subgenus Camponotus because our research group is focusing on this subgenus in this and several other projects. In April 2022, a somewhat contiguous genome of C. pennsylvanicus (a member of the subgenus Camponotus) was reported in a preprint (Faulk, 2022); our genome is assembled into scaffolds ~10× more contiguous than that of this reported C.

| Genome assembly and annotation
We assembled the Camponotus sp.
First, we used Canu v1.9 (Koren et al., 2017) to de novo assemble the PacBio long reads. Second, we used the Hi-C sequence data to scaffold the initial assembly using the 3D-DNA pipeline (Dudchenko et al., 2017;Durand et al., 2016). All commands for the assembly, annotation, and all further analyses are documented on GitHub (github. com/jdman they/campo notus_genomes1). We quality checked the genome assembly for potential contamination with BlobTools v. We used a multistep process to annotate transposable elements (TEs) and repetitive elements in the Camponotus sp.
(1-JDM) genome: (1) identify de novo repeats and over-represented sequences, (2) manually curate repetitive elements, and (3) mask the genome with these elements to create a TE and repetitive element summary file.
First, we removed any RepeatModeler output sequences ≥98% identical to RepBase sequences. Second, we used BLAST and bedtools (Camacho et al., 2009;Quinlan & Hall, 2010) to extract genomic regions matching repetitive elements as well as 1000 bp flanking sequences. We used these extracted sequences to develop consensus sequences for novel TEs using the following steps: (1) alignment using MAFFT (Katoh & Standley, 2013)  . We used these initial MAKER predictions to train SNAP and Augustus (Korf, 2004;Stanke & Waack, 2003). Lastly, we used the models trained in SNAP and Augustus in a second iteration of MAKER to predict gene models in the Camponotus sp.
To align putative homologues between the four ant species, we used T-Coffee (Notredame et al., 2000). T-Coffee translates nucleotide sequences, aligns them using several alignment algorithms, takes the averaged best alignment of all alignments, and back translates the protein alignments to provide a nucleotide alignment for each gene. Before the final back-translating, we used trimAl (Capella-Gutiérrez et al., 2009) to remove gaps in the protein alignments.
We tested each gene for selection using gene-wide and branchspecific tests for selection in CODEML (Yang, 1997). After correct- ing significance values for multiple testing using the Benjamini and Hochberg (1995) method, we removed any alignments with evidence for selection. We then extracted and concatenated four-fold degenerate sites from the alignments (N = 806,844) using custom R scripts and the R packages "Biostrings" and "seqinr" (Charif & Lobry, 2007;Pagès et al., 2017). With this alignment of four-fold degenerate sites, we identified an appropriate model of sequence evolution using jModelTest (Darriba et al., 2012) and used the GTR + I model of sequence evolution in PhyML (Guindon et al., 2010) to estimate a phylogenetic tree.
To put the evolution of the CDS four-fold degenerate sites in a timed evolutionary context, we downloaded a recent phylogenomic tree of formicine ants (Blaimer et al., 2015) and pruned the tree to the four representative lineages covered by our CDS downloads and novel assembly using the R package "ape" (Paradis et al., 2004) using four species as representatives of those lineages: Camponotus (Myrmentoma) hyatti Emery, 1893, Nylanderia dodo (Donisthorpe, 1946), Formica neogagates Viereck, 1903, and Lasius niger. We used the Camponotus-specific branch length of the fourfold degenerate sites tree along with divergence time estimates from Blaimer et al. (2015) to obtain an estimate of Camponotus-specific mutation rates. (SRX5650044). With our newly generated data and the downloaded data, we trimmed adapters and quality filtered the raw sequencing data using the bbduk.sh script of the bbmap package (Bushnell, 2014). We then aligned the filtered data to the de

| Blochmannia genome assemblies and annotation
With the raw sequencing data, we used the MinYS pipeline (Guyomar et al., 2020) to assemble Blochmannia genomes for each sample.
MinYS used samples mixed with host and bacterial DNA in a pipeline that allows targeted assembly of bacterial genomes. First, it maps metagenomic reads to a reference genome using BWA. Here, we  (Wick et al., 2015), regions with multiple paths merged by coverage, and output in FASTA format. This process assembled a circular genome for each of the samples in our study. We also downloaded the sequence and annotation of the Blochmannia endosymbiont of Camponotus floridanus for use as an outgroup (NC_005061.1; Gil et al., 2003). We used the NCBI prokaryotic genome annotation pipeline (Tatusova et al., 2016) to annotate genes in each of the Blochmannia genomes.

| Genetic diversity and demography
We estimated genetic diversity for each individual in two ways. First, we estimated observed heterozygosity simply as the proportion of bi-allelic to total genotyped sites (both invariant and variant) for each individual. Because sequencing depth has the potential to impact estimates of genetic diversity, we also used the program ROHan (Renaud et al., 2019), which uses a Bayesian framework to estimate rates of heterozygosity while accounting for sequencing depth and per-base quality scores. We found the two estimates to be highly MSMC output is presented relative to a species' generation time and mutation rate. We used the mutation rate calculated from the de novo genome assembly as described above. Because there are no good estimates of generation times in Camponotus ants, we used a conservative proxy for generation time used in other studies: double the age of sexual maturity (Nadachowska-Brzyska et al., 2015). In Camponotus, previous studies have suggested that the earliest age of queens producing winged reproductives is a minimum of 2 years following colony formation, with the first winged individuals overwintering until the third year (Fowler, 1986;Pricer, 1908). As such, we used 3 years as the age of reproductive maturity and 6 years as

| Tests for selection
We used the HyPhy software package (Pond & Muse, 2005) to test for selection in the Blochmannia genes in a phylogenetic framework. We tested for positive selection using aBSREL (M. D. Smith et al., 2015) and we tested for shifts in selection strength across the phylogeny using RELAX . We ran aBSREL in exploratory mode where all branches are tested for positive selec-

| Genetic drift
For each species' Blochmannia genomes, we estimated the rate of nonsynonymous substitutions per site (K a ) relative to the rate of synonymous substitutions per site (K s ) using the R package "SeqinR" (Charif & Lobry, 2007). Generally, the K a /K s ratio is indicative of the strength of selection in coding genes; in a particular gene, we may expect values much less than one under the effects of purifying selection and values greater than one due to positive selection. However, we expect very low genome-wide K a /K s ratios due to selection maintaining function of most genes in the genome ); in species with smaller effective population sizes, we expect increased genetic drift and reduced efficacy of selection that may result in accumulation of slightly deleterious mutations and higher estimates of genome-wide K a /K s ratios . As such, we used genome-wide K a /K s ratios as a proxy for the strength of genetic drift in each species.

| Gene loss
We tested for gene loss in all Blochmannia genomes assembled for this study. First, we performed an all-to-all protein BLAST (blastp) of all amino acid sequences from coding genes annotated from all samples. From the BLAST analysis results, we tabulated a gene presence/absence matrix for each Blochmannia genome (N = 607 unique coding genes identified from all samples).

| Co-analyses of Camponotus and Blochmannia data
To estimate correlations between host and endosymbiont traits while accounting for the evolutionary history of the samples, we analyzed all host and Blochmannia traits in the context of phylogenetic independent contrasts (PICs). We estimated PICs for each trait in the R package "ape" (Paradis et al., 2004) which uses the method of Felsenstein (1985).
This PIC method assumes Brownian motion of trait evolution and transforms the sampled trait data into statistically independent values (contrasts) that may be used in regressions (Felsenstein, 1985).

| Evolutionary rates
We explored evolutionary rates in Blochmannia genomes in multiple ways. First, we examined variation in Blochmannia gene phylogenies relative to the host species tree. To do this, we calculated the Kuhner and Felsenstein (1994); KF94) distance between the host species tree and Blochmannia gene trees, implemented in the R package "ape" (Paradis et al., 2004). Second, we explored rates of evolutionary change in a phylogenetic context by measuring relative branch lengths in the host species tree versus the Blochmannia gene trees.
We calculated these rates of evolutionary change in three groups: (1) subgenus Camponotus, (2) subgenus Tanaemyrmex excluding C. ocreatus because it is on a long branch by itself, and (3) the combined group of the subgenera Camponotus and Tanaemyrmex. Third, we measured nucleotide percent identity for all gene alignments, excluding indels, in the same three groups as aforementioned. Fourth, for each sampled individual, we measured the correlation between host and endosymbiont root-to-tip distance in the species tree phylogenies.

| Demographic influences on drift, selection, and gene loss
For each of the six ingroup species (i.e., excluding C. ocreatus), we measured the association between host population sizes and (1) changes in selection strength in Blochmannia genes, (2) Blochmannia genome-wide K a /K s ratios, (3) number of complete genes found in each Blochmannia genome, and (4) Blochmannia genome size.

| Camponotus reference genome characteristics and molecular clock
Our de novo Camponotus sp.
(1-JDM) reference genome was highly contiguous (contig L50 ~ 500 kbp) with a small number of scaffolds composing the majority of the assembly (scaffold L90 ~ 3.58 Mbp, N90 = 29; see Table 3). Overall, we had 31 scaffolds greater than 2 Mbp. Although there are no karyotypes for North American species in the subgenus Camponotus, there are estimates for Camponotus ligniperda (Latreille, 1802) (haploid N = 14), C. japonicus Mayr, 1866 (N = 13 or 14), and C. obscuripes Mayr, 1866 (N = 14) from the eastern Palearctic (Hauschteck, 1983;Imai, 1969;Imai & Yosida, 1964). As such, it appears we generated a genome with the contiguity of about two scaffolds per chromosome. While we did have additional signal in the Hi-C contacts to additionally scaffold the genome (Figure 2a), we chose to be conservative and only link genomic regions where we were confident of the signal (Figure 2a).
The difficulty in fully scaffolding the genome may relate to the repetitive nature of the genome; the genome averaged 24.7% repetitive element content with many large portions of the genome exhibiting greater than 70% repetitive content ( Figure 2b). Overall, about 13% of genomic windows contained more than 60% repetitive content. A large proportion of the repetitive content was DNA transposons, both previously described and those manually curated for this study. The repetitive landscape is consistent with other ant species exhibiting "islands" of extreme repetitive content in a background of lower genomic repetitive content (Schrader et al., 2014).

Coding gene content is heterogeneous across the genome and is
negatively correlated with both repetitive element content and GC% in 100 kbp sliding windows (Figure 2b,c). BUSCO results suggest our genome is nearly complete and representative of other hymenopterans, containing 98% complete genes and 1.2% fragmented genes of the 4415 hymenopteran near-universal single-copy orthologs (Table 4). Using the four-fold degenerate sites from the genome's CDS regions, we estimated a substitution rate of 1.983877 × 10 −9 substitutions/site/year.

| Phylogenomics
We estimated an ant host species tree using 4770 "gene trees" estimated in 50 kbp sliding windows across the genome. Both methods we used to create the species tree-maximum clade credibility and ASTRAL-identified an identical topology (Figure 1) while each node had 100% support in ASTRAL analyses (Figure 1).  The Blochmannia species tree estimated from 507 gene trees identified a strongly supported phylogeny with a nearly identical topology to the host species tree (Figure 1). The only differences were some relationships between individuals within species (colored orange in Figure 1). In contrast to varying proportions of gene trees matching species tree relationships in the ant hosts, a majority of Blochmannia gene trees matched relationships of the species tree (67%-99% support for each node). We measured the KF94 distance between each Blochmannia gene tree and the host species tree to identify if any regions of the Blochmannia genome were relatively discordant from the host phylogenomic signal. In general, the phylogenetic concordance, as measured by the KF94 metric, was consistent across the entire Blochmannia genome (Figure 3a).

| Blochmannia genome sizes, gene composition, rates of molecular evolution
The Blochmannia genomes assembled here largely varied per subgenus. In the subgenus Camponotus, the genomes varied in size from Root-to-tip Distances

PIC Host Root-to-tip Distances
The Blochmannia genomes contained between 576 and 601 genes, and in total across all genomes, 607 unique coding genes were annotated. In total, 65 genes exhibited variable presence/absence among the samples sequenced here (Figure 4). Of those 65 genes, 37 exhibited phylogenetic signal of gene loss.
The Blochmannia genes evolved at rates ~20-30× faster than the rate of evolution of the ant hosts, with slight variation across the Blochmannia genome (Figure 3b). Additionally, it appeared that Blochmannia genes in ant hosts of the subgenus Camponotus had a slightly slower rate of evolution than those of the ant host subgenus Tanaemyrmex (Figure 3b). If we use the Camponotus rate of molecular evolution to put Blochmannia rates in a timed absolute context, the mean Blochmannia gene evolution rate is about 5.474 × 10 −8 substitutions/site/year (range = 1.454 × 10 −8 to 1.256 × 10 −7 substitutions/ site/year). Species tree root-to-tip distances for host and endosymbionts (trees in Figure 1) showed a significant positive correlation ( Figure 3D), suggestive of a genome-wide molecular evolution association between hosts and their endosymbionts.
Blochmannia gene identity within host subgenera was generally consistent across the endosymbiont genome, suggestive of similar evolutionary forces acting across most of the genome at the taxonomic scale of host clades ( Figure 3C). We also tried to identify relative rates of evolution and percent sequence identity in intergenic regions. To do this, we performed whole-genome alignments using progressiveMauve (Darling et al., 2010).
However, endosymbiont intergenic sequences were so divergent between host subgenera, and in some cases, between host species, that we were unable to recover any high-quality alignments in these regions (e.g., large nonoverlapping sections in these regions of the alignments). Needless to say, the rates of evolution in these intergenic regions are likely much higher than the genic rates in Figure 3b.
We found a strong positive association between Blochmannia genome-wide K a /K s ratios and number of Blochmannia genes with re-

| Impacts of host demography on endosymbiont evolution
Contemporary estimates of host effective population sizes ranged from ~5000 to 50,000 and, with a couple of exceptions, were largely consistent within species (Figure 6). Variation in demographic histories within species may be indicative of variation in among population gene flow (i.e., a lack of panmixia), and therefore, the overall demographic trends for each individual should be interpreted with this in mind. Overall, however, harmonic mean population size through the last 200,000 years was highly correlated with observed heterozygosity for each individual (r = 0.850, p << .001), and suggests that the MSMC population size estimates reflect population history, even if not simply population size trends (e.g., variance in estimates due to differential population structure). As such, we looked for correlations between endosymbiont traits and host population sizes using these harmonic mean population size estimates.
In positive selection tests, 19 Blochmannia genes showed evidence for selection. These signatures of positive selection appeared somewhat randomly in the phylogeny (not shown). We also tested for shifts in selection strength (i.e., intensified or relaxed) among the host lineages for all Blochmannia genes. We found a positive relationship between host population size estimates and number of endosymbiont genes with shifts toward intensified selection pressures ( Figure 7). In contrast, we found no relationship between number of genes with relaxed selection strength and host demography (not shown). As previously mentioned, some gene loss in endosymbiont genomes exhibited phylogenetic signal (Figure 4). Despite this, we found no evidence for a relationship between host population size and gene loss in endosymbiont genomes (Figure 7c). Additionally, we found no significant associations between host population sizes and either Blochmannia genome-wide K a /K s ratios (Figure 7b) or Blochmannia genome size (Figure 7d).

| DISCUSS ION
We sequenced 17 carpenter ant hosts and their Blochmannia endosymbionts to address questions about host demography impacts on endosymbiont evolution. We added a whole-genome resource for one species in the subgenus Camponotus and more than doubled the number of publicly available Blochmannia full-genome sequences.
With these resources, we investigated questions related to the (1) codiversification of hosts and endosymbionts, (2) molecular evolution of endosymbionts, and (3)

| Codiversification of carpenter ant hosts and Blochmannia endosymbionts
Using whole-genome sequencing of both carpenter ant hosts and their bacterial endosymbionts, we identified generally strict codiversification ( Figure 1). There was some phylogenetic incongruence between host and endosymbiont trees among individuals within species, but all species-level relationships were completely congruent. These patterns are consistent with expectations of co-speciation between hosts and vertically transmitted endosymbionts; similar evidence of codiversification between hosts and endosymbionts has been found in bivalves (Distel et al., 1994), weevils (Toju et al., 2013), flies (Chen et al., 1999;Hosokawa et al., 2012), cockroaches (Clark et al., 2001;Lo et al., 2003), aphids (Clark et al., 2000), psyllids (Thao et al., 2000), and previous studies in carpenter ants (Degnan et al., 2004;Sauer et al., 2000). Generally, previous studies investigating codiversification have inferred phylogenies using one or a few molecular markers; in contrast, by sequencing full genomes for both hosts and endosymbionts, we were able to obtain strongly supported species trees as well as estimate variation in lineage sorting across Blochmannia genes ( Figure 3a) with phylogenetic statistics. Here, we expected one of two pat-

| Rates of molecular evolution in Blochmannia endosymbionts
We found that Blochmannia genes evolved at a rate ~ 30× faster than the host genome ( Figure 3). In addition, intergenic regions were so divergent across lineages that we were not able to align them properly. This endosymbiont-host relative evolution rate is similar to the level reported in Buchnera bacterial endosymbionts of aphids (~36×) by Moran et al. (1995). On an absolute scale, we estimated  (Clark et al., 1999).
Relatively, fast evolution rates are expected in endosymbionts because of their life histories; insect endosymbionts' asexuality and propensity to undergo regular bottlenecks because of their mode of inheritance lead to small effective populations sizes and relatively fast evolution (Mira & Moran, 2002;Wernegreen, 2002). As such, endosymbionts also have faster evolutionary rates compared with their free-living relatives, including increased rates of evolution at nonsynonymous coding sites (Brown & Wernegreen, 2016;Degnan et al., 2004;Moran, 1996). Because Blochmannia endosymbionts are asexual and likely to have small population sizes, they may undergo rapid genetic drift and experience accelerated molecular evolution (Pettersson & Berg, 2007;Rispe & Moran, 2000;Woolfit & Bromham, 2003). Additionally, even with small population sizes, obligate endosymbionts may still be under very strong within-host selective pressures, further accelerating their molecular evolution (Perreau et al., 2021).
Endosymbiont molecular evolution rates may vary somewhat across the genome and may have host-lineage-specific rates of molecular evolution ). Indeed, we found that relative rates of molecular evolution varied somewhat across Blochmannia genomes (Figures 3b,c). Additionally, we found that lineage-specific rates of Blochmannia molecular evolution were correlated with host rates of evolution ( Figure 3d). These results are similar to those found in Camponotus and Blochmannia using a small genetic dataset (two host genetic loci and four Blochmannia genetic loci) (Degnan et al., 2004) and suggest that molecular evolution rates-while much faster in Blochmannia-are correlated between carpenter ant hosts and Blochmannia endosymbionts at genomewide scales. Correlated rates of molecular evolution between hosts and endosymbionts have also been demonstrated in (1) aphids and their Buchnera endosymbionts (Arab & Lo, 2021) and (2) cockroaches and their Blattabacterium endosymbionts (Arab et al., 2020).
Overall, our results corroborate previous evidence that endosymbionts have faster rates of evolution relative to both their hosts and to their free-living bacterial relatives, even when evolutionary rates are correlated between hosts and their endosymbionts.

| Does host demography shape endosymbiont evolution?
Because population genomic processes are influenced by effective population size, and endosymbiont effective population size is intrinsically linked with host effective population sizes (Mira & Moran, 2002;Wernegreen, 2002), we may have the expectation that host demographic patterns partially influence endosymbiont molecular evolution. Here, we investigated whether host demography influenced four factors of endosymbiont genome evolution: (1) natural selection, (2) genetic drift, (3) genome size, and (4) patterns of gene loss.
We found no relationship between host demography and both signatures of positive selection and relaxation of selection strength in Blochmannia genes. In contrast, we found a positive relationship between host population sizes and shifts toward intensified selection pressures in Blochmannia genes (Figure 7a). In endosymbionts in general, we may expect relaxed selection relative to patterns in free-living bacteria (Wernegreen, 2002). Indeed, selection is often identified in insect endosymbionts, but generally, only in a small fraction of genes (Alleman et al., 2018;Chong et al., 2019;Williams & Wernegreen, 2012). Based on our results (Figure 7a), it appears that shifts in selection pressures may at least in part be influenced by host demographic processes.
We also tested for an effect of host demography on patterns of endosymbiont genetic drift, gene loss, and overall genome size.
Here, we found no relationship between host population sizes and these characteristics of endosymbiont genome evolution (Figure 7).
We initially anticipated that endosymbiont genetic drift, and associated gene loss and genome size evolution, would occur faster in endosymbionts with small host effective population sizes. While this was not the case with the entire dataset (Figure 7), the host species with the smallest estimated population sizes-C. laevissimus Mackay, 2019-showed the (1) highest rate of endosymbiont genetic drift as measured by genome-wide K a /K s ratios, (2) most endosymbiont genes with shifts toward relaxed selective pressures relative to other endosymbiont lineages, and (3) lowest Blochmannia gene count (Table 1).
Additionally, we found that about half of the gene loss was phylogenetically informative (Figure 4). This suggests relatively random patterns of gene loss in the phylogeny; most gene losses lacking phylogenetic signal were singleton gene losses (Figure 4). This is consistent with previous research in Blochmannia endosymbionts identifying lineage-specific gene loss largely due to relaxed selection constraints and genetic drift (Williams & Wernegreen, 2015).
Similarly, in cockroach Blattabacterium endosymbionts, Kinjo et al. (2021) found that gene loss was lineage-specific, and that some genes showed parallel gene loss across multiple Blattabacterium lineages.
While we did not find a relationship between host demography and the evolution of Blochmannia genome sizes, we identified associations between a combination of relaxed selection strength, genome-wide genetic drift, and Blochmannia genome sizes ( Figure 5).
Higher genome-wide K a /K s ratios were strongly associated with smaller Blochmannia genome sizes ( Figure 5b). These results suggest that genetic drift, or possibly a combination of genetic drift and relaxed selection strength, is shaping genome size reduction in the Blochmannia genomes sampled here. This negative association between genome-wide K a /K s ratios and genome size is like that identified by  across 42 free-living and symbiotic pairs of bacteria and suggests a general relationship between the relative rate of nonsynonymous genetic changes and genome size reduction.
Overall, we found that host demography is associated with shifts in selection strength in Blochmannia genomes, but not associated with several other aspects of Blochmannia molecular evolution. As such, we may infer that either (1) our small sample sizes (number of species) may be precluding us from identifying weak correlations between host demography and Blochmannia molecular evolution, or (2) host effective population sizes may not directly reflect endosymbiont effective population sizes, leading to a lack of or weak relationship between host population sizes and endosymbiont molecular evolution. If different host species or populations have varied patterns of endosymbiont transmission population bottlenecks (Mira & Moran, 2002) or endosymbiont within-host selection (Perreau et al., 2021), we may expect a decoupling of host and endosymbiont effective population sizes.

| CON CLUS IONS
We used whole-genome sequencing of both carpenter ant hosts and their Blochmannia endosymbionts to investigate the influence of host demography on symbiont molecular evolution. We identified strict codiversification of Camponotus hosts and their Blochmannia endosymbionts. Blochmannia genes are evolving about 30× faster than host genomes, with relatively consistent evolutionary rates across the Blochmannia genome. We found that some, but not all, patterns of natural selection in Blochmannia genomes were in part shaped by host demographic history. Blochmannia genome size evolution was not associated with host demography but was associated with genome-wide estimates of genetic drift and number of genes with relaxed selection pressures.

ACK N OWLED G M ENTS
This work was supported by Texas Tech University startup funds to JDM. JDM thanks John Jisha for getting him interested in Camponotus more than a decade ago with a month-long field trip.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data associated with this manuscript may be found in the fol-